双氢青蒿素对Ⅱ型胶原诱导的类风湿关节炎大鼠的抗炎和软骨修复作用

Anti-inflammatory Effects and Cartilage Repair of Dihydroartemisinin for Rats with Rheumatoid Arthritis Induced by TypeⅡCollagen

【目的】探讨双氢青蒿素对Ⅱ型胶原诱导的类风湿关节炎(RA)大鼠的抗炎作用和软骨修复作用。【方法】将75只健康6周龄雄性SD大鼠随机分为假手术组,模型组,双氢青蒿素低、中、高剂量组,每组15只。除假手术组外,其他各组大鼠均给予Ⅱ型胶原诱导构建RA模型。成功造模后,双氢青蒿素低、中、高剂量组大鼠分别对应给予双氢青蒿素6.25、12.5、25 mg/kg灌胃处理,1次/d,连续给药2周。给药结束后,检测骨指标(骨体积分数、骨表面积/骨体积、骨小梁厚度、骨小梁数量);采用苏木素-伊红(HE)染色法观察踝关节组织病理学表现、阿利新蓝染色法观察踝关节软骨组织蛋白多糖的表达,采用抗酒石酸酸性磷酸酶(TRAP)染色法观察踝关节骨组织破骨细胞情况;采用酶联免疫吸附分析(ELISA)检测外周血中肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-10、IL-17、IL-23的含量;采用蛋白免疫印迹法检测踝关节组织成骨标记物成骨细胞特异性转录因子Osterix(Osx),Ⅰ型胶原蛋白α1链(COL1A1)和骨钙素(OC)的表达;采用实时定量聚合酶链反应(RT-PCR)检测踝关节组织骨吸收功能相关基因破骨细胞相关受体(OSCAR)和TRAP的表达情况。【结果】与假手术组比较,模型组大鼠骨体积分数,骨小梁厚度、骨小梁数量显著降低,骨表面积/骨体积显著升高(均P<0.05),踝关节组织病理学表现可见滑膜增生,炎性细胞浸润,软骨损伤严重,软骨组织基本无蛋白多糖表达,外周血中炎症相关因子TNF-α、IL-17、IL-23和IL-10含量均显著升高(P<0.05),踝关节组织Osx、COL1A1、OC的相对表达水平均显著降低(P<0.05),OSCAR、TRAP的mRNA表达水平均显著升高(P<0.05)。给予双氢青蒿素干预后,RA大鼠上述指标均得到明显改善。【结论】双氢青蒿素可以通过抑制炎症反应、促进软骨修复治疗Ⅱ型胶原诱导的大鼠RA。

Objective To explore the anti-inflammatory effects and cartilage repair of dihydroartemisinin(DHA)for rats with rheumatoid arthritis(RA)induced by typeⅡcollagen.Methods Seventy-five healthy 6-week-old male SD rats were randomly divided into sham-operation group,model group,low-,middle-and high-DHA groups,15 rats in each group.Apart from the sham-operation group,RA model was established in the other groups by typeⅡcollagen induction.After successful modeling,the low-,middle-and high-DHA groups were given intragastric administration of DHA at the dosage of 6.25,12.5,25 mg/kg,respectively,once every day,the treatment lasting 2 weeks.After medication,the bone indexes[bone volume/total volume,bone surface/bone volume,trabecular bone thickness,trabecular bone number]were measured,the changes of histologic features of ankle tissue was observed by hematoxylin-eosin(HE)staining,the proteoglycan expression in cartilage tissue was observed by Alcian blue staining,the production of osteoclasts in ankle tissue was observed by tartrate resistant acid phosphatase(TRAP)staining,the levels of tumor necrosis factorα(TNF-α),interleukin(IL)-17,IL-23and IL-10 in peripheral blood were detected by enzyme-linked immunosorbent assay(ELISA),the expression levels of osteogenic markers[osterix(Osx),collagen typeⅠalpha1(COL1A1)and osteocalcin(OC)]in ankle tissue were detected by Western blotting assay,and the m RNA expression levels of osteoclast associated receptor(OSCAR)and TRAP in ankle tissue were detected by real-time polymerase chain reaction(RT-PCR).Results Compared with the sham-operation group,bone volume/total volume,trabecular bone thickness and trabecular bone number were decreased,and bone surface/bone volume was increased in the model group,the pathological features of ankle tissue showed synovial hyperplasia,inflammatory cell infiltration,serious cartilage injury,no proteoglycan expression in cartilage tissue,the contents of inflammation-related factor TNF-α,IL-17,IL-23,IL-10 in peripheral blood were enhanced(P<0.05),the relative expression levels of Osx,COL1A1,OC in ankle tissue were lowed(P<0.05),and the m RNA expression levels of OSCAR,TRAP in ankle tissue were raised(P<0.05).After intervention of DHA,the above indexes in RA rats were improved.Conclusion DHA is effective for treatment of typeⅡcollagen-induced RA in rats by inhibiting inflammation reactions and promoting cartilage repair.